A Steric-Inhibition Model for Regulation of Nucleotide Exchange Via the Dock180 Family of Gefs

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Provided by Elsevier - Publisher Connector Current Biology, Vol. 15, 371–377, February 22, 2005, ©2005 Elsevier Ltd All rights reserved. DOI 10.1016/j.cub.2005.01.050 A Steric-Inhibition Model for Regulation of Nucleotide Exchange via the Dock180 Family of GEFs

Mingjian Lu,1 Jason M. Kinchen,3 Kent L. Rossman,4 and cell migration [1, 11]. These include both the cata- Cynthia Grimsley,1,2 Matthew Hall,5 John Sondek,4 lytic Docker domain that is located within the C-terminal Michael O. Hengartner,3 Vijay Yajnik,5 half of Dock180 and mediates nucleotide exchange on and Kodi S. Ravichandran1,* Rac and an approximately 350 amino acid N-terminal 1Beirne Carter Center for Immunology Research region that contains an SH3 domain. To better define Department of Microbiology and the molecular features of this N-terminal region of 2 Department of Pharmacology Dock180, we generated various Dock180 mutants (Fig- University of Virginia ure S1 in the Supplemental Data available with this arti- Charlottesville, Virginia 22908 cle online). Biochemical characterization of these mu- 3 Institute of Molecular Biology tants revealed two ELMO-interacting sites within this University of Zurich region. One interaction is mediated by the binding of 8057 Zurich, Switzerland the Dock180 SH3 domain to the PxxP motif of ELMO 4 Department of Pharmacology (Figure 1A). In addition, a second region adjacent to the University of North Carolina SH3 domain also binds ELMO and was disrupted by a Chapel Hill, North Carolina 27599 G171E mutation in Dock180 (Figure 1B; see below for 5 Gastrointestinal Unit description of G171E). It is noteworthy that these two Massachusetts General Hospital types of interaction are independent of each other. Harvard Medical School While characterizing these Dock180 mutants, we un- Boston, Massachusetts 02114 expectedly observed that the SH3-domain-deleted Dock180 was more active in a phagocytosis assay than wild-type Dock180 (Figure 1C, lanes 6 and 8), suggesting Summary that the SH3 domain of Dock180 might play a basal inhibitory role. One possible mechanism for such SH3- CDM (CED-5, Dock180, Myoblast city) family members mediated negative regulation is through binding to an- have been recently identified as novel, evolutionarily other region (or other regions) of Dock180. Consistent conserved guanine nucleotide exchange factors (GEFs) with this notion, the isolated SH3 domain (Dock1-83) or for Rho-family GTPases [1–7]. They regulate multiple a slightly larger N-terminal fragment of Dock180 (Dock1- processes, including embryonic development, cell mi- 161), coprecipitated with Dock (⌬SH3) (Figure 1D and gration, apoptotic-cell engulfment, tumor invasion, data not shown). No interaction was detectable between and HIV-1 infection, in diverse model systems [4, 6, the SH3 domain and the C-terminal proline-rich tail of 8–16]. However, the mechanism(s) of regulation of Dock180 (data not shown). When other regions of Dock180 CDM proteins has not been well understood. Here, our were examined, the Dock1–161 fragment or the isolated studies on the prototype member Dock180 reveal a steric-inhibition model for regulating the Dock180 SH3 domain readily bound the catalytic Docker domain family of GEFs. At basal state, the N-terminal SH3 (Figure 1E, lane 4; Figure S2) Intriguingly, the SH3: domain of Dock180 binds to the distant catalytic Docker interaction does not seem to be a typical Docker domain and negatively regulates the function SH3:PxxP binding; first, the W45A mutation within the of Dock180. Further studies revealed that the SH3: Dock180 SH3 domain, which abolished the domain’s Docker interaction sterically blocks Rac access to the binding to the PxxP motif of ELMO (Figure 1A), had no Docker domain. Interestingly, ELMO binding to the effect on binding to the Docker domain (Figure 1E, lane SH3 domain of Dock180 disrupted the SH3:Docker in- 5); second, disrupting the single PxxP motif within the teraction, facilitated Rac access to the Docker domain, Docker domain did not affect the Docker:SH3 interaction and contributed to the GEF activity of the Dock180/ (Figure 1E, lane 6). ELMO complex. Additional genetic rescue studies in We then aligned the amino acid sequences from the C. elegans suggested that the regulation of the SH3 domains of CDM family members and compared Docker-domain-mediated GEF activity by the SH3 do- the consensus sequence with those from other SH3 main and its adjoining region is evolutionarily conserved. domains. We identified a conserved isoleucine residue This steric-inhibition model may be a general mecha- (I32 of Dock180) within CDM family members; in most nism for regulating multiple SH3-domain-containing other SH3 domains, this residue is a lysine or glutamate Dock180 family members and may have implications (Figure S3). Of note, this I32 residue resides in the ligand for a variety of biological processes. binding RT loop of the SH3 domains. Mutating this isoleucine residue to lysine (I32K) completely abrogated Results and Discussion the SH3:Docker interaction (Figure 1F, lane 3). We then tested whether the I32K mutation introduced An Inhibitory Interaction between the SH3 into the full-length Dock180 could functionally mimic the and Docker Domains of Dock180 effects of deleting the SH3 domain. Remarkably, the Previous studies showed a functional requirement for I32K mutation enhanced the activity of Dock180 in at least two regions of Dock180 during phagocytosis phagocytosis in a manner similar to that of the SH3- domain deletion (Figure 1C, lanes 7 and 8). When one *Correspondence: [email protected] considers that the I32K mutation disrupted the SH3 Current Biology 372

Figure 1. An Inhibitory Interaction between SH3 and Docker Domains of Dock180 (A) The PxxP motif of ELMO binds to the SH3 domain of Dock180. GST-Dock1-161 or its W45A or I32K mutant version was transfected into 293T cells with either full-length ELMO-GFP or its PxxP-deletion mutant. After GST precipitation, the bound proteins were analyzed by immunoblotting. (B) The second interaction between Dock180 and ELMO and its disruption by G171E mutation. Flag-Dock180 or one of its mutants was coexpressed with ELMO-GFP in 293T cells. Pre-cleared cell lysates were subject to anti-Flag precipitation. The bound proteins were analyzed by immunoblotting. The ELMO1-629, which does not bind Dock180 [1], was used as a negative control. (C) Phagocytosis assay. LR73 cells were transfected with wild-type Flag-Dock180 or one of its mutants together with either GFP (lanes 2–4) or ELMO-GFP (lanes 6–8). The percentage of GFP-positive cells with engulfed particles was determined in a flow-cytometry-based engulfment assay. The error bar represents the standard deviation. (D) The SH3 domain of Dock180 binds to the SH3 domain-deleted Dock180 mutant. Flag-Dock (⌬SH3) was coexpressed in 293T cells with either GST or GST-Dock1-161. Cleared cell lysates were precipitated with anti-Flag and immunoblotted with the indicated antibodies. (E) Interaction between the SH3 domain and the Docker domain of Dock180. Wild-type or mutant Flag-Docker domain and the indicated GST- Dock1-161 or its W45A mutant were expressed in 293T cells, precipitated with anti-Flag antibody, and immunoblotted with the indicated antibodies. The PP→AA mutation disrupted the single PxxP motif within the Docker domain. (F) I32K mutation within the SH3 domain disrupts SH3 interaction with the Docker domain. Flag-Docker domain was cotransfected with GST- Dock1-161 or its I32K mutant into 293T cells, precipitated with anti-Flag antibody, and immunoblotted with the indicated antibodies. Regulation of Dock180-Family GEFs 373

binding to Docker, these data support the hypothesis disrupted its binding to both the Docker domain and that the SH3:Docker interaction inhibits the functional ELMO (Figures 1A and 1F). Collectively, these results activity of Dock180. suggest that the binding of the PxxP motif of ELMO to the SH3 domain of Dock180 can disrupt the SH3:Docker interaction. SH3 Domain Blocks Rac Access Consistent with the above observations, the binding to the Docker Domain of an ELMO fragment carrying the PxxP motif enhanced Previous studies have shown that the binding of nucleo- Rac binding to Dock180 (Figure 3C, lanes 2 and 5) to tide-free Rac to the Docker domain is critical for Rac an extent roughly equal to that observed with the Dock activation by the Dock180/ELMO complex [1]. We hy- (⌬SH3) mutant (lanes 1 and 5). In contrast, coexpression pothesized that the SH3 domain might exert its inhibitory of ELMO (lanes 3 and 6) did not affect the binding of effect by blocking the access of Rac to the Docker nucleotide-free Rac to the Dock (W45A) mutant. Of note, domain. Interestingly, the Dock (⌬SH3) mutant associ- here we used the W665A mutant of ELMO to avoid the ated with nucleotide-free Rac significantly better than ELMO PH-domain-mediated contribution to Rac binding wild-type Dock180 (Figure 2A). A similar result was seen [18]. Similarly, ELMO interaction with Dock2 contributed with another CDM family member, Dock2 [17], wherein to enhanced Rac binding (Figure 2B). Collectively, these deletion of the N-terminal region resulted in better Rac data suggested that the PxxP motif of ELMO binding to binding (Figure 2B). Moreover, addition of nucleotide- the SH3 domain of CDM proteins could relieve the SH3- free Rac inhibited the SH3 domain binding to the Docker domain-mediated steric hindrance. domain (Figure 2C, lanes 2 and 3), suggesting that the We next assessed the functional relevance of the Dock- SH3 domain and nucleotide-free Rac competitively bind SH3:ELMO-PxxP interaction. In a fluorescence-based GEF the Docker domain. Under the same conditions, the GDP assay, in which the association between Dock180 and bound Rac, which does not bind the Docker domain with ELMO was primarily mediated via the SH3:PxxP interac- high affinity, did not reduce the SH3:Docker interaction tion, the Dock180/ELMO complex showed 2-fold en- (Figure 2C, lanes 5 and 6). hancement in GEF activity over Dock180 alone (Figure Compared with wild-type Dock180, the Dock (⌬SH3) 3D, curves e and i). This enhancement was similar to that mutant also showed increased basal Rac activation in observed with wild-type Dock180 (curves d and g). In cells (increase of 1.6-fold Ϯ 0.2-fold, p Ͻ 0.05, n ϭ 3) contrast, the SH3 mutant Dock (W45A) failed to syner- (Figure 2D). Deleting the SH3 domain from Dock180 also gize with ELMO (W665A) in the in vitro GEF assay (data rendered it more active in the in vitro GEF assay (Figure not shown). When examined in the Dock180/ELMO- S4). Recent studies suggest that wild-type Dock4 can mediated, Rac-dependent Transwell migration of LR73 promote Rac-GTP loading within cells when it is overex- cells, the Dock (W45A) mutant failed to synergize with pressed (M.H. and V.Y., unpublished data). Also, com- ELMO in promoting cell migration, whereas wild-type pared to wild-type Dock4, Dock4 in which the SH3 do- Dock180 promoted migration (Figure 3E, lanes 5 and 6). main was deleted led to increased Rac-GTP loading in At the level of the whole organism, we assessed the cells (increase of 1.7-fold Ϯ 0.3-fold, Figure 2D). These effect of disrupting the PxxP:SH3 interaction in C. elegans results suggested that the SH3 domain of CDM proteins gonadal distal-tip cell (DTC) migration. During C. elegans has a negative regulatory role with regard to the Docker- development, the distal-tip cell migrates in a stereotypi- domain-mediated Rac activation and that the removal cal U-shaped pattern to define the shape of the adult of such inhibition may be required for the optimal activa- hermaphrodite gonad. In worms with null mutations in tion of this family of proteins. ced-12 or ced-5, the DTC frequently mismigrates with extra or wrong turns. This phenotype can be rescued, ELMO Binding to the SH3 Domain Relieves respectively, by transgenic expression of wild-type ced- the Inhibitory SH3:Docker Interaction 12 or ced-5. However, a ced-12 transgene with muta- Compared with Dock180 alone, the Dock180/ELMO tions of its PxxP motif was less efficient in rescuing the complex binds to the nucleotide-free Rac more effi- DTC-migration defects in CED-12 null animals (Table 1). ciently and shows higher Rac-GEF activity [1, 10, 18]. Similarly, a ced-5 (⌬SH3) mutant was also less efficient Based on previous studies [1, 2, 10, 11, 19, 20], ELMO in rescue of DTC mismigration in CED-5 null worms was considered as a prime candidate for disrupting the (Table 1). Taken together, these data suggested that the SH3:Docker interaction via binding to the Dock180 SH3 Dock-SH3:ELMO-PxxP interaction is functionally rele- domain (Figure 3A). Consistent with this model, coex- vant in vitro and in vivo and that a requirement for this pression of ELMO reduced the amount of Dock1–161 interaction is evolutionarily conserved. that coprecipitated with the Docker domain (Figure 3B, lanes 1 and 3). In contrast, the Dock1–161:Docker interaction was not reduced by an ELMO mutant that lacks The Region Adjacent to the SH3 Domain Also the PxxP motif (lanes 1 and 5). Similarly, the binding of Affects the GEF Activity of Dock180 the Docker domain to the Dock1–161 (W45A) mutant The regulation of the basal activity of Dock180 by its was not reduced by coexpression of ELMO (lanes 2 N-terminal region was also supported by another set of and 4). The Dock180 SH3 domain did not appear to observations. In Drosophila Myoblast city, a single point simultaneously bind the Docker domain and ELMO be- mutation within the region adjacent to its SH3 domain cause no detectable ELMO coprecipitated with the SH3 (corresponding to G171E mutation in Dock180; Figure domain bound to the Docker domain (data not shown). S5) resulted in dorsal-closure and myoblast-fusion de- Furthermore, the I32K mutation within the SH3 domain fects comparable to those of Mbc null alleles [9]. Inter- Current Biology 374

Figure 2. SH3 Domain Blocks the Access of Rac to the Docker Domain (A) Deletion of the SH3 domain promotes the binding of nucleotide-free Rac to Dock180. Flag-Dock180 or its SH3-deletion mutant was transfected into 293T cells with either GST or GST-Rac. The total cell lysates were subjected to GST precipitation in the presence of 10 mM EDTA and immunoblotted with the indicated antibodies. (B) Deletion of the N-terminal region of Dock2 or association with ELMO enhances nucleotide-free Rac binding to Dock2. A Dock2 (⌬N) construct [17] or wild-type Dock2 with or without ELMO was expressed in 293 T cells, and lysates were precipitated with coexpressed GST- Rac (in the presence of EDTA) and assessed for coprecipitation of Dock2 or its ⌬N mutant. It is notable that the Dock2 (⌬N) construct was poorly expressed in comparison to wild-type Dock2. After densitometry, the signals for Rac-associated Dock2 were normalized against protein expression in the total lysates, and the signal intensity for wild-type Dock2 alone was arbitrarily set to 1. (C) Nucleotide-free Rac competes with Dock1-161 in binding to the Docker domain. Flag-Docker was expressed and precipitated from transfected 293T cells. Precipitates were then incubated with bacterially produced Dock1-161 or Rac (1 ␮g of each) as indicated. Normalized quantitation of the intensity of the GST-Dock1-161 bands is indicated below each band. The results are representative of two independent experiments. LC indicates the light chain of antibody. (D) SH3-domain-deleted Dock180 and Dock4 are more active in Rac activation within cells. 293T cells were cotransfected with Rac and wild- type Dock180 or its ⌬SH3 mutant. The intracellular Rac-GTP level was assessed, in triplicates, by precipitation with GST-CRIB beads and immunoblotting for Rac (upper panel). Rac-GTP levels were normalized against the expression level of Rac and Dock180 proteins (lower left panel). Results from similar experiments comparing wild-type and ⌬SH3 versions of Dock4 were also presented (lower-right panel). The error bars denote one standard deviation. estingly, G171E mutation within Dock180 resulted in an ELMO (Figure 1B). These combined effects might ex- approximately 50% reduction in the basal in vitro GEF plain the severe phenotypes associated with this muta- activity (Figure 3D, curves d and e). This G171E mutation tion in Drosophila. When tested in CED-5-deficient also disrupted the second interaction of Dock180 with worms, the Dock (G171E) mutant was completely defec- Regulation of Dock180-Family GEFs 375

Figure 3. ELMO Relieves the SH3-Mediated Inhibition of Dock180 (A) Model figure depicting the binding of ELMO to the SH3 domain of Dock180. This binding disrupts the SH3:Docker interaction and allows Rac better access to the Docker domain. (B) Binding of the PxxP motif of ELMO to the SH3 domain of Dock180 disrupts the SH3:Docker interaction. Flag-Docker domain was coexpressed in 293T cells with wild-type or mutant GST-Dock1-161 in the presence or absence of ELMO-GFP. Cell lysates were subject to anti-Flag- antibody precipitation, and the coprecipitation of Dock1-161 in the presence or absence of ELMO was assessed by immunoblotting. After densitometry, the signal for GST-Dock1-161 in the precipitates was normalized against its expression level in the total cell lysates, and the GST-Dock1-161 value was set at 1 for the purpose of comparison. The results are representative of three independent experiments. (C) ELMO enhances the binding of nucleotide-free Rac to wild-type Dock180 but not its W45A mutant. Indicated Dock180 or its mutant was expressed in 293T cells with ELMO532-727 (W665A)-GFP (denoted as ELMO*). Cell lysates were subject to precipitation with bacterially produced GST-Rac in the presence of EDTA. (D) In vitro GEF assay. Flag-Dock180 or its G171E mutant was expressed in 293T cells either alone or together with ELMO-GFP or its W665A mutant as indicated. The proteins were precipitated with anti-Flag antibody and eluted with Flag peptide. The eluted proteins were quantitated after anti-Flag blotting, and equal amounts of Dock180 protein were used in a mant-GDP fluorescence-based in vitro GEF assay. The observed mant-GDP dissociation-rate constants (␬obs) were indicated. (E) Transwell migration assay. Wild-type Dock180 or its W45A mutant was transfected into LR73 cells with ELMO (W665A)-GFP. The relative cell migration of transfected cells through a Transwell was scored. Current Biology 376

Table 1. The SH3 and Adjacent Regions of CED-5/Dock180 and the PxxP Motif of CED-12 Influence DTC Migration In Vivo Genotype Mismigration (Percent) n (A) Effect of SH3 Deletion of CED-5 or PxxP Mutation of CED-12 on DTC Migration DTC Mismigration in ced-5- and ced-12 Null Worms Is Rescued by ced-5 and ced-12 Wild-type 0 204 ced-5(n1812) 35 314 ced-12(k149) 36 305 ced-5(n1812); opEx732 [Peft-3::ced-5(WT)] 5 173 ced-12(k149); opEx872 [Peft-3::ced-12(WT)::yfp] 9 302 Partial Rescue of Mismigration in ced-12 Null Worms by the PxxP Mutant of ced-12 ced-12(k149); opEx874 [Peft-3::ced-12(AxxA)::yfp] 18 305 ced-12(k149); opEx873 [Peft-3::ced-12(AxxA)::yfp] 21 302 Partial Rescue of Migration Defects in ced-5 Null Worms by ced-5(⌬SH3) ced-5(n1812); opEx723 [Peft-3::ced-5(⌬SH3)] 22 305 ced-5(n1812); opEx725 [Peft-3::ced-5(⌬SH3)] 23 326 ced-5(n1812); opEx726 [Peft-3::ced-5(⌬SH3)] 24 315 (B) Effect of G171E Mutation of Dock180 on Rescuing DTC-Migration Defects in CED-5-Deficient Worms Partial Rescue of DTC-Migration Defects in ced-5 Null Worms by Wild-Type Dock180 ced-5(n1812); opEx763 [Peft-3::Dock180(WT)] 24 310 No Rescue of DTC-Migration Defects in ced-5 Null Worms by Dock(G171E) ced-5(n1812); opEx379 [Peft-3::Dock180(G171E)] 44 302 ced-5(n1812); opEx371 [Peft-3::Dock180(G171E)] 53 405 ced-5(n1812); opEx382 [Peft-3::Dock180(G171E)] 50 304 During C. elegans development, the distal-tip cell (DTC) migrates in a stereotypical U-shaped pattern to define the shape of the adult hermaphrodite gonad (wild-type). In worms with null mutations in ced-5 or ced-12, the DTC frequently migrates incorrectly with extra or wrong turns (mismigrated). The ced-5(n1812) mutant or the ced-12(k149) was made transgenic for the indicated constructs. The percentage of gonadal arms with migration defects was scored in multiple independent transgenic lines for each construct. All lines were coinjected with the Plim_7::GFP marker for visualizing the gonadal arms. All of the CED-5, Dock180 and CED-12 constructs were expressed under the Peft-3 promoter. tive in rescuing the DTC-migration defect, and in fact it the regulation of GEF activity of several CDM family enhanced the DTC mismigration (Table 1). Taken to- members, and this model has implications for multiple gether, these data suggest that the SH3 domain and its biological processes. adjoining region critically regulate the GEF activity of Dock180 and that the ELMO binding to the N-terminal Supplemental Data region of Dock180 may alleviate the inhibitory SH3: Supplemental Experimental Procedures, three figures, and a table are available with this article online at http://www.current-biology. Docker interaction and fully activate Dock180. com/cgi/content/full/15/4/371/DC1/. Our data suggest a steric-inhibition model for regulation of the GEF activity of CDM family members. In this Acknowledgments model, the SH3 domain binding to the catalytic Docker domain blocks the access of Rac to the Docker domain; We thank Michiyuki Matsuda for the original Dock180 and the Dock2 this inhibition is relieved, at least in part, by the binding plasmids and members of the Ravichandran lab for helpful sugges- of the PxxP motif of ELMO to the SH3 domain and tions during this work. This work was supported by National Insti- tutes of Health grants GM-64709 (to K.S.R.) and DK63933 (to V.Y.), allows Rac to have better access to the Docker domain. an Infectious Diseases Training grant from the National Institutes Although the SH3 domain in the basal state negatively of Health (to C.G.), and grants from the Swiss National Science regulates the Rac-GEF activity of Dock180, it provides Foundation, The Ernst Hadorn Foundation, and the European Union one of the two contact sites for ELMO and thereby posi- (FP5 project APOCLEAR) (to M.O.H.). tively affects Rac-GEF activity of the Dock180/ELMO complex. The latter effect was best revealed in the Received: November 17, 2004 Revised: December 15, 2004 C. elegans in vivo rescue studies involving CED-5 mu- Accepted: December 20, 2004 tants lacking the SH3 domain. Interestingly, a self-inhibi- Published: February 22, 2005 tory mechanism is also utilized by certain conventional DH-PH-containing Rho-GEFs to regulate their basal ca- References talytic activity [21]. The SH3 domain is present in the five mammalian CDM family members, with at least four 1. Brugnera, E., Haney, L., Grimsley, C., Lu, M., Walk, S.F., Tosello- of them shown to bind ELMO [1, 2, 11, 13]. The data Trampont, A.C., Macara, I.G., Madhani, H., Fink, G.R., and Ravi- chandran, K.S. (2002). Unconventional Rac-GEF activity is medi- presented here on Dock180, Dock2, and Dock4, along ated through the Dock180-ELMO complex. Nat. Cell Biol. 4, with the worm rescue studies using mutants of Dock180 574–582. and CED-5, support a novel steric-inhibition model for 2. Sanui, T., Inayoshi, A., Noda, M., Iwata, E., Stein, J.V., Sasazuki, Regulation of Dock180-Family GEFs 377

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